Natural killer T (NKT) cells comprise a specialized subset of T lymphocytes that express cell surface markers characteristic of NK cells. The ability of these cells to produce cytokines, activate cells of both the innate and adaptive immune responses, and recognize antigen in the context of CD1d molecules strongly suggest that NKT cells play a pivotal role in anti-tumor immunity. However, anti-tumor effects mediated by these cells may be compromised because cancer patients have a reduction in both NKT cell number and function. Therefore, we hypothesize that adoptive transfer of effector NKT cells will be an effective therapeutic strategy to enhance cancer immunotherapy. Our lab has previously developed MHC-lg and CD1d-lg-based artificial Antigen Presenting Cells (aAPC), which can facilitate the induction and expansion of primary T and NKT cells. In this proposal, we will further modify this CD1d-lg based aAPC to stimulate and analyze different NKT cell subsets in vitro. In addition we will evaluate the in vivo anti-tumor activity of the different NKT cell subsets in a human melanoma/SCID model. Collectively, these studies provide a novel approach to study the in vitro expansion as well as the in vitro and in vivo function of NKT cell subsets for potential use in adoptive cancer immunotherapy is presented. The information gained in the proposed studies will serve to help understand and design NKT cell based approaches to enhance current immunotherapeutic treatment strategies for cancer. A major goal in cancer immunotherapy is to generate an effective anti-tumor immune response. This application represents a novel approach using artificial Antigen Presenting Cells, aAPC, to study activation and expansion of NKT cells to enhance the efficiency of cancer immunotherapy.